RESEARCH ARTICLES

Cite as: T. Nakamura et al., Science

10.1126/science.aaw0843 (2020).

Noncanonical transnitrosylation network contributes to

synapse loss in Alzheimer’s disease
Tomohiro Nakamura1,2*†, Chang-ki Oh1,2†, Lujian Liao1‡, Xu Zhang1,2, Kevin M. Lopez2, Daniel Gibbs3, Amanda
K. Deal1, Henry R. Scott1, Brian Spencer3, Eliezer Masliah3§, Robert A. Rissman3,4, John R. Yates III1, Stuart A.
Lipton1,2,3*
1Departments of Molecular Medicine and Neuroscience, and Neuroscience Translational Center, The Scripps Research Institute, La Jolla, CA 92037, USA.
2Neurodegenerative Disease Center, Scintillon Institute, San Diego, CA 92121, USA. 3Department of Neurosciences, University of California, San Diego, School of Medicine,
La Jolla, CA 92093, USA. 4VA San Diego Healthcare System, San Diego, CA, USA.
*Corresponding author. Email: tnakamura@scripps.edu (T.N.); slipton@scripps.edu (S.A.L.)
†These authors contributed equally to this work. ‡Present address: Shanghai Key Laboratory of Regulatory Biology and Shanghai Key Laboratory of Brain Functional

Downloaded from http://science.sciencemag.org/ on December 4, 2020

Genomics, School of Life Sciences, East China Normal University, Shanghai 200241, China. §Present address: National Institute on Aging/NIH, Bethesda, MD, USA.

We describe mechanistically-distinct enzymes, i.e., a kinase, a guanosine triphosphatase and a ubiquitin

protein hydrolase, which function in disparate biochemical pathways, that can also act in concert to
mediate a series of redox reactions. Each enzyme manifests a second, noncanonical function –
transnitrosylation – triggering a pathological biochemical cascade in Alzheimer’s disease (AD). The
resulting series of transnitrosylation reactions contributes to synapse loss, the major pathological
correlate to cognitive decline in AD. We conclude that enzymes with distinct primary reaction mechanisms
can form a completely separate network for aberrant transnitrosylation. This network operates in the post-
reproductive period, so natural selection against such abnormal activity may be decreased.

Excessive nitric oxide (NO) can engender nitrosative stress in
the nervous system, contributing to neurodegenerative dam-
age (1, 2). In Alzheimer’s disease (AD), oligomerized amyloid-
β peptide (Aβ), neuronal hyperexcitability, and neuroinflam-
mation associated with aging can each trigger NO generation
and resultant S-nitrosylation (donation of an NO group most
likely as a nitrosonium cation intermediate, NO+) to specific
cysteine thiol (or more properly thiolate anion, -S−) (3–14).
One such cysteine is located on dynamin-related protein 1
(Drp1), a guanosine triphosphatase (GTPase) that mediates
mitochondrial fission (15). This posttranslational redox reac-
tion, forming SNO-Drp1, hyperactivates the enzyme, leading
to mitochondrial fragmentation and bioenergetic compro-
mise, with consequent synapse loss, in part because of the
great demand for energy during neurotransmission (15, 16).
Because reduction in synapse number is closely correlated
with cognitive decline in AD (17, 18), this nitrosylation reac-
tion may contribute to disease pathogenesis (4). S-Nitrosyl-
ated cyclin-dependent kinase 5 (Cdk5) (19) acts to pass NO to
Drp1 to form SNO-Drp1 via a reaction termed transnitrosyla-
tion (20). However, additional members of this pathological
cascade are yet to be determined. Characterizing this aber-
rant series of reactions may further reveal its contribution to
the pathogenesis of AD and possible therapeutic implications
of the pathway. We used both in vitro and in vivo models of
AD as well as human AD postmortem brains to show that en-
zymes with disparate catalytic activities from different
biochemical pathways can form a transnitrosylation cascade
that contributes to synapse loss in AD. We demonstrate that
the deubiquitinylating enzyme, Uch-L1 (Ubiquitin carboxyl-
terminal hydrolase isozyme-L1), contributes to a transnitro-
sylation cascade leading to synaptic damage in AD that in-
volves transfer of a NO group from Uch-L1 to Cdk5 and then
to Drp1. Although other potential members of the cascade re-
main to be determined, our results show pathways mediating
a series of aberrant transnitrosylation reactions may be im-
portant in the etiology of neurodegenerative disorders.
S-Nitrosylation of Uch-L1 in cell lines and AD
transgenic mice by biotin switch and mass spectrometry
We examined upstream events to S-nitrosylation of Cdk5
(forming SNO-Cdk5), which in turn leads to SNO-Drp1
formation and consequent Aβ-related synaptic spine loss in
AD (15, 20). The ubiquitin hydrolase Uch-L1 can potentiate
cyclin-dependent kinase activity by an unknown reaction
mechanism not involving its ubiquitin hydrolase activity (21),
and we found that this mechanism involves transnitrosyla-
tion to Cdk5.
Aberrant S-nitrosylation of a number of proteins that
affect their canonical enzymatic activity has been demon-
strated in AD and other neurologic diseases (4), including
members of the ubiquitin pathway such as the E3 ligase
Parkin and the ubiquitin hydrolase Uch-L1 (22–24). However,
we discovered that aberrant and seemingly unrelated

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S-nitrosylated enzymes are linked by multiple transnitrosyla-
tion steps between them that result in a wholly disparate cas-
cade of biochemical pathological activity. We exposed human
neural SH-SY5Y cells to a physiological NO donor, S-nitro-
socysteine (SNOC). We then made cell lysates and analyzed
them for S-nitrosylated Uch-L1. Using the biotin-switch
method (25), we found evidence for the formation of SNO-
Uch-L1 under these conditions (Fig. 1A). Additionally, endog-
enous NO, generated by activation of stably-expressed neu-
ronal NO synthase (nNOS) in human embryonic kidney
(HEK) cells (designated HEK-nNOS cells), also resulted in
formation of SNO-Uch-L1 (Fig. 1B); nNOS activation by the
calcium ionophore A23187 led to a ~2-fold increase in the rel-
ative abundance of SNO-Uch-L1 in replicate experiments. The
absence of ascorbate in the reaction mixture abrogated detec-
tion of the increase in SNO-Uch-L1 via the biotin-switch assay
because, under these conditions, NO was not removed and
replaced by biotin (Fig. 1, A and B). Of potential pathological
relevance, we found increased SNO-Uch-L1 compared to wild-
type (WT) littermates in cerebrocortices of AD transgenic
mouse models overexpressing human amyloid precursor pro-
tein (hAPP-J20 and Tg2576), which produce Aβ oligomers
(Fig. 1C and fig. S1). The very high abundance of Uch-L1 in
the brain might favor its S-nitrosylation, not necessarily re-
quiring a catalyst for the reaction.
We used site-directed mutagenesis in HEK-293 cells to in-
vestigate which cysteine residue was S-nitrosylated in Uch-
L1. Mutation of Cys152 decreased formation of SNO-Uch-L1,
indicating that it is a major site of S-nitrosylation after expo-
sure to exogenous SNOC (Fig. 1D) or endogenous NO in HEK-
nNOS cells (Fig. 1E). Additionally, the NOS inhibitor NG-nitro-
l-arginine (NNA) prevented S-nitrosylation of WT Uch-L1,
confirming that NO was indeed the reactant (Fig. 1E). We
used mass spectrometry-based S-nitrosoproteomics analysis
to confirm the predominant site of S-nitrosylation as Cys152
(Fig. 2, A and B, and table S1). Moreover, we identified an
acidic (Glu)-basic (Arg) region as a candidate motif for S-ni-
trosylation surrounding the critical Cys residue in the atomic
resolution model of Uch-L1 (Fig. 2C) (12, 26, 27).
Effect of S-nitrosylation on Uch-L1 deubiquitinylation
activity in cell-based models and AD transgenic mice
To determine the effect of formation of SNO-Uch-L1, we
monitored Uch-L1 binding to ubiquitin and Uch-L1 deubiqui-
tinylation activity. Exposure to SNOC decreased Uch-L1 asso-
ciated with ubiquitin and decreased deubiquitinylation
activity in both recombinant and cell-based assays (fig. S2, A
to C). As a control, non-nitrosylatable mutant Uch-L1(C152S)
was not affected by SNOC. Additionally, in the hAPP-J20
transgenic mouse model of AD, we observed a decrease in the
active form of Uch-L1, i.e., Uch-L1 bound to ubiquitin on im-
munoblots (fig. S2D). Conversely, treatment with ascorbate
First release: 3 December 2020 www.sciencemag.org
(to reductively remove NO from SNO-protein) resulted in an
increase in the active form of Uch-L1, consistent with increased
ubiquitinylation activity due to denitrosylation of Uch-L1.
Decreased Uch-L1 activity is observed in human brains
with AD as well as in AD mouse models (28, 29). Hence,
SNO-Uch-L1 formation in AD brain, with consequent inhibi-
tion of Uch-L1 activity, might contribute to this effect. Over-
expression of Uch-L1 decreases Aβ production, increases
activity of protein kinase A (PKA) and cAMP response
element-binding protein (CREB), and improves synaptic
function and memory performance in AD transgenic mice
(29–31). Therefore, Uch-L1 activity appears to be important
in the pathogenesis of the disease.
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Oligomeric Aβ triggers SNO-Uch-L1 formation and
synapse loss in AD models in vitro and in vivo
We reasoned that if the therapeutic action of Uch-L1 overex-
pression was mediated by decreasing endogenous Aβ produc-
tion, then the pathological effects on synapses of adding
exogenous Aβ oligomers might not be greatly affected by
Uch-L1 overexpression. On the other hand, if the beneficial
effect of Uch-L1 occurred, at least in part, through another
function of the enzyme, we should observe this therapeutic
action in the presence of exogenous Aβ oligomers. Indeed,
S-nitrosylation of Uch-L1 occurred in a neuronal cell-based
model upon exposure to Aβ oligomers–incubation of primary
rat cerebrocortical cultures in 500 nM Aβ1-42 oligomers
resulted in formation of SNO-Uch-L1 as determined by the
biotin-switch assay (Fig. 3A and fig. S3A).
Additionally, after exposure of rat cerebrocortical neurons
to 500 nM Aβ1-42 oligomers, approximately half of the
dendritic spines were lost (Fig. 3, B and C) (15, 20, 32–34);
loss of synaptic spines was dependent on NO, as preincuba-
tion in NNA abrogated the effect (15, 20). Transfection with
non-nitrosylatable mutant Uch-L1, but not with WT Uch-L1,
decreased the Aβ-induced loss of dendritic spines in a statis-
tically significant manner, although there was also a trend
toward improvement with WT Uch-L1 (Fig. 3C and fig. S3B).
This trend with WT Uch-L1 may reflect overexpression of WT
protein, which can act as a sink for NO (allowing substantial
WT protein to remain un-nitrosylated) and therefore poten-
tially offer some benefit (15). However, non-nitrosylatable
mutant protein is more effective and showed statistically sig-
nificant synaptic protection.
To determine if S-nitrosylated Uch-L1 also contributes in
vivo to synapse loss, we used the hAPP-J20 transgenic mouse
model of AD, which overexpresses oligomeric Aβ in the brain
(35). We generated lentiviral vectors expressing tdTomato
fluorescent protein and Uch-L1 (WT or C152S), and injected
them into the dentate gyrus of hAPP-J20 AD mice (fig. S4, A
and B). Two months later, we evaluated the histological ef-
fects on presynaptic integrity. Quantitative unbiased confocal
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immunohistochemistry of hippocampal sections with an an-
tibody to the presynaptic marker synaptophysin (SY38) (36,
37) revealed that expression of Uch-L1(C152S), but not that of
WT Uch-L1, significantly increased SY38 signal compared to
that obtained from vehicle-treated hAPP-J20 AD mice (Fig. 3,
D and E). Although overexpression of WT protein afforded a
trend toward presynaptic protection, probably because it
acted as a sink for NO (15), only non-nitrosylatable mutant
protein resulted in statistically significant protection of the
presynaptic marker. These findings are consistent with the
notion that SNO-Uch-L1 contributes to synapse loss in AD
models. Because synapse loss is a close neuropathological
correlate to cognitive decline in human AD, this finding sup-
ports biological relevance to the disease state.
Transnitrosylation from Uch-L1 to Cdk5 to Drp1
Aβ oligomer-induced S-nitrosylation of Drp1 results in
mitochondrial fragmentation, bioenergetic compromise, and
consequent synapse loss (15, 16, 38). Studies with purified
recombinant proteins support direct transfer of an NO group
(representing transnitrosylation) to Drp1 from SNO-Cdk5
(20), although the upstream effector linking Aβ-induced NO
generation to S-nitrosylation of Cdk5 was unknown. Given
our new evidence for S-nitrosylation of Uch-L1 in AD trans-
genic mouse brain and the fact that Uch-L1 potentiates
cyclin-dependent kinase activity by a reaction mechanism
not involving its ubiquitin hydrolase activity (21), we tested
whether Uch-L1 might contribute to an NO-mediated path-
way to synapse loss, leading to a series of transnitrosylation
steps among these enzymes from diverse biochemical path-
ways (NO group transfer from Uch-L1 to Cdk5 to Drp1).
We performed kinetic, cell-based S-nitrosylation assays to
monitor possible transfer of an NO group from SNO-Uch-L1
to Cdk5 and then to Drp1. Using the biotin-switch assay, we
found evidence for formation of both SNO-Uch-L1 and SNO-
Cdk5 in HEK-293 cells within 10 min of exposure to SNOC;
with increasing formation of SNO-Drp1 by 25 min, concur-
rently with a relative decrease in the amount of SNO-Cdk5
(Fig. 4, A to D). The fact that RNAi depletion of Uch-L1, com-
pared to control siRNA (siCTL), partially prevented for-
mation of SNO-Cdk5 under these conditions at 10 min is
consistent with the notion that transnitrosylation occurs
from upstream SNO-Uch-L1 to Cdk5 (Fig. 4, A to C). Similarly,
RNAi depletion of Uch-L1 also partially prevented the subse-
quent formation of SNO-Drp1 (Fig. 4D). Although RNAi
depletion of Uch-L1 decreased SNO-Cdk5 and SNO-Drp1 gen-
eration, it did not eliminate it, presumably because SNOC is
a non-enzymatic donor of NO, producing super-physiological
amounts of protein S-nitrosylation that would be expected to
continue undeterred when competing with transnitrosyla-
tion among proteins. To clarify further, this experiment indi-
cates that donation of NO from SNOC to SNO-Uch-L1
First release: 3 December 2020 www.sciencemag.org
occurred, but SNOC also S-nitrosylated Cdk5 and Drp1. The
fact that knockdown of Uch-L1 decreased to a statistically sig-
nificant degree the formation of SNO-Cdk5 (at 10 min) and
SNO-Drp1 (at 10 min and even more so at 25 min) under these
conditions argues that SNO-Uch-L1 triggered downstream
transnitrosylation reactions (20, 39).
By graphing the difference of relative SNO-Cdk5 and SNO-
Drp1 concentrations, as calculated from control (siCTL) val-
ues compared to those in cells lacking Uch-L1 (siUch-L1), we
can subtract the effect of S-nitrosylation due to SNOC and
quantify the effect of transnitrosylation between the enzymes
(Fig. 4, C and D). The temporal delay in appearance of SNO-
Drp1 relative to SNO-Cdk5 is consistent with the notion that
SNO-Cdk5, in turn, may transnitrosylate Drp1.
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To characterize this putative transnitrosylation pathway
from SNO-Cdk5 to Drp1, we used in vitro nitrosylation assays.
We transfected SH-SY5Y neural cells with WT Uch-L1 or mu-
tant Uch-L1(C152S), each tagged with V5 (derived from a
small epitope found in paramyxovirus proteins), prepared
cell lysates, and performed immunoprecipitation with anti-
body to V5. Immunoprecipitated Uch-L1 protein (WT or
C152S mutant) was then exposed to SNOC and incubated for
a sufficient period of time to ensure near total dissipation of
NO from SNOC (20, 39). The WT or C152S mutant Uch-L1 im-
munoprecipitates were then added to cell lysates containing
hemagglutinin (HA)-tagged Cdk5 (Fig. 5, A to E). We moni-
tored possible transnitrosylation reactions by probing for for-
mation of SNO-V5-Uch-L1, SNO-HA-Cdk5, and endogenous
SNO-Drp1 by biotin-switch assay. SNO-Cdk5 and SNO-Drp1
formed in the WT Uch-L1-immunoprecipitated lysates after
SNOC exposure but significantly less was found in immuno-
precipitates from mutant Uch-L1(C152S) (Fig. 5, A to E). Alt-
hough immunoprecipitate experiments do not rule out the
possibility that additional members of the cascade participate
in these transnitrosylation reactions, they support participa-
tion of Uch-L1 and Cdk5 in the reaction sequence leading to
the generation of SNO-Drp1.
The relative degree of transnitrosylation of Drp1 resulting
from S-nitrosylated Uch-L1 was dependent on the amount
of endogenous Cdk5. When Cdk5 was depleted by immunopre-
cipitation, the amount of transnitrosylated SNO-Drp1 de-
creased (Fig. 5, F to I). The degree of immunodepletion of
endogenous Cdk5 corresponded with the decrement in SNO-
Drp1 formation in the presence of WT Uch-L1 but not in the
presence of C152S mutant Uch-L1 (Fig. 5G). This finding
confirms that SNO-Cdk5, rather than SNO-Uch-L1, predomi-
nantly mediates transnitrosylation to Drp1 under these condi-
tions. This type of experiment is also consistent with the notion
that these enzymes transnitrosylate with a preference of one
substrate over another. Mechanistically, the particular envi-
ronment of the thiolate anion can kinetically favor one NO
donor over another.
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 In cell lysates expressing either HA-Cdk5 or V5-Uch-L1,
we found transnitrosylation from SNO-Uch-L1 to Cdk5, and
then to Drp1, but lack of NO group transfer from SNO-Cdk5
to Uch-L1 (Fig. 6A). These experiments, although lacking
formal derivation of rate constants, turnover number (kcat)
or substrate affinity (inversely related to Km) to support an
enzymatic process (40), do provide additional evidence for
specific transnitrosylation reactions because of relative
substrate preference. Thus, the entire pathway of NO transfer
from SNO-Uch-L1 to SNO-Cdk5 to SNO-Drp1 had to be
present in order to observe significant SNO-Drp1 formation
under these conditions. Nonetheless, other proteins present
in the immunoprecipitates may participate in this series of
transnitrosylation events. To show more definitively that
SNO-Uch-L1 directly transfers NO to Cdk5, we performed
an in vitro assay with purified recombinant proteins.
SNO-Uch-L1 could directly transfer an NO group to Cdk5
(fig. S5).
Because of differences in methods between standard im-
munoblotting and the biotin-switch method, one cannot
compare the density of the bands representing the total input
protein and the S-nitrosylated protein for evaluation of the
absolute concentration of the SNO-protein. However, we can
compare the ratio of SNO-protein to total (input) protein to
obtain the relative amount of S-nitrosylated protein, and we
can then compare this ratio across various preparations (20,
39, 41). Therefore, more critical to the overall hypothesis of a
series of transnitrosylation events than the actual magnitude
of an individual SNO-protein band generated is the fact that
we could detect significant differences in their relative abun-
dance. This ratio concept is also amenable to precise quanti-
tative analysis of electron transfer and associated energetics
of transnitrosylation reactions under steady-state conditions,
as described below.
Nernst equations to predict transnitrosylation
pathways and Gibbs free energy at steady state
In general, biological redox reactions often do not go to com-
pletion and depend on the relative redox potential of the pair
of proteins that interact. Thus, SNO-protein bands observed
in biotin-switch assays are not expected to be completely
abated when individual upstream or downstream compo-
nents are disrupted. Accordingly, we devised a quantitative
method based on the Nernst equation to characterize these
transnitrosylation reactions thermodynamically (20, 39). For
this analysis, we make the assumption that these reactions go
to steady state, as might be reasonably expected to occur in a
chronic disease. The Nernst equation quantifies the electro-
motive force for net electron movement between a redox pair.
To calculate the difference in the electromotive force (ΔE) be-
tween two redox pairs, e.g., for transnitrosylation between
Uch-L1 and Cdk5, we calculated the difference in the values
First release: 3 December 2020 www.sciencemag.org
of their respective Nernst equations:
0′ 0′ RT  [ UchL1SNO ][ Cdk5red ] 
EUchL1 − ECdk5 =
− ⋅ ln  74.02 mV
=
zF  [ UchL1 ][ Cdk5 ] 
 red SNO 
(1)
 [ UchL1red ][ Cdk5SNO ] 
∆G 0′ =
− RT ⋅ ln  −18.35 kJ/mol
=
 [ UchL1 ][ Cdk5 ] 
 SNO red 
(2)
where “SNO” represents the S-nitrosylated (or oxidized) pro-
tein and “red” represents the chemically reduced protein, as
obtained from densitometric analysis of redox immunoblots
(Fig. 6B). Because the Nernst equation uses log values, the
difference in Nernst equations represents the natural log of
the ratio of effective concentrations of the reduced and oxi-
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dized forms of the protein. Thus, the absolute concentrations
of the reduced protein and oxidized (in this case, S-nitrosyl-
ated) protein are not needed for the evaluation of the blots or
for the resulting calculation; only the relative ratio is re-
quired. Hence, we can treat the reactions quantitatively with-
out actually knowing the exact protein concentrations.
Using this method, we found that transnitrosylation from
SNO-Uch-L1 to Cdk5 is predicted to be energetically very fa-
vorable under steady-state conditions in intact cells. Evidence
for this was obtained by obtaining the relative redox potential
for the reaction (Eq. 1, ΔE0′ = 74.02 ± 4.60 mV [n = 4]), which
in turn allowed us to calculate the associated change in Gibbs
free energy (Eq. 2, ΔG0′ = −18.35 ± 1.14 kJ/mol [n = 4]), pre-
dicting the reaction will proceed spontaneously. Our previous
results indicated that the subsequent transnitrosylation from
SNO-Cdk5 to Drp1 was also energetically favorable (20). Col-
lectively, these findings lead us to propose that an NO group
is transferred from SNO-Uch-L1 to Cdk5, which leads to sub-
sequent transfer from SNO-Cdk5 to Drp1 in an energetically-
favorable manner.
S-Nitrosylation of Uch-L1 in human AD brains
To assess the potential pathophysiological relevance of our
findings in human AD, we obtained human postmortem
brain tissue (table S2). Abundance of SNO-UchL1 was signif-
icantly increased in human brains with advanced stages of
AD compared to that in control brains (Fig. 7A and fig. S6).
Amounts of SNO-Uch-L1 were extremely low by these meth-
ods in control human brains, consistent with the notion that
S-nitrosylation of Uch-L1 represents an aberrant nitrosyla-
tion event occurring in the diseased state only. To determine
whether accumulation of SNO-Uch-L1 in human AD brain
might be of pathophysiological significance, we calculated
the ratio of SNO-Uch-L1 (by biotin-switch assay) to total Uch-
L1 (as quantified from immunoblots), using methods previ-
ously described (20, 39, 41). In human AD brain this ratio was
comparable to or greater than that encountered in our
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neuronal cell-based models exhibiting Aβ/SNO-Uch-L1—
induced dendritic spine loss (Figs. 3A and 7B) or in the brains
of hAPP-J20 AD mice (Figs. 1C and 7B), which exhibit synapse
loss (42). Taken together, these findings indicated that
the aberrant transnitrosylation cascade from SNO-Uch-L1
to Cdk5 to Drp1 may contribute to the synaptic pathology
observed in AD (Fig. 7C).
Discussion
The abundance of Uch-L1 protein is decreased in human
brains with sporadic AD (28). On the other hand, overexpres-
sion of Uch-L1 delays AD progression in vivo in animal mod-
els (29). This is thought to occur, at least in part, because of
decreased Aβ production (30, 31). In the continued presence
of exogenous oligomeric Aβ, however, Uch-L1 still modulates
synaptic damage in AD models, implicating a second mecha-
nism of Uch-L1 action. Along these lines, in in vivo experi-
ments, expression of mutant Uch-L1(C152S) in mice is
neuroprotective (43), implicating the possible importance of
redox reactions at the critical Cys residue 152 of Uch-L1.
We found that Cys152 of Uch-L1 is S-nitrosylated in human
AD brain to a similar degree as in our cell-based and animal
models of AD. This reaction forms SNO-Uch-L1, which in
turn, transnitrosylates Cdk5, an enzyme that functions
in neuronal maturation and migration, among other pro-
cesses. Next, SNO-Cdk5 transnitrosylates Drp1, a GTPase that
mediates mitochondrial fission. Not only do these transnitro-
sylation reactions occur in cell-based immunoprecipitates,
but also in in vitro transnitrosylation assays using puri-
fied/recombinant enzymes, showing that the donation of
the NO group proceeds directly from one protein to another.
Hence, the data show that there is overall donation of an NO
group via a series transnitrosylation reactions from Uch-L1
to Cdk5 to Drp1 (Fig. 7C). The S-nitrosylation of Drp1 triggers
excessive mitochondrial fission/fragmentation with conse-
quent bioenergetic loss and synaptic damage (15, 20). This
finding may influence progression of AD, as synapse loss is
a major neuropathological correlate of cognitive decline in
AD (17, 18).
The proteins that participate in this transnitrosylation
cascade (Uch-L1/Cdk5/Drp1) also function in totally dispar-
ate pathways with different mechanisms of enzymatic
action (ubiquitin hydrolase, kinase, and GTPase, respec-
tively). One reason that this second noncanonical function of
transnitrosylation may occur is that the catalytic sites of
many types of enzymes contain cysteine residues, and their
thiol groups (in fact, thiolates) are nucleophiles that can
perform a reversible nucleophilic attack on the nitroso nitro-
gen via an S-nitrosylation reaction scheme (44–46). This
reaction is particularly favored under conditions of high
NO-induced nitrosative stress, as found in neurodegenerative
diseases associated with aging.
First release: 3 December 2020 www.sciencemag.org
In conclusion, we have discovered sequential NO transfer
among unrelated enzymes that is not accounted for by the
canonical catalytic activity originally discovered for the en-
zymes. This concept of “noncanonical transnitrosylation net-
works” may represent a set of biochemical reactions not
currently enveloped in standard bioinformatics packages
such as gene ontology (GO) enrichment or KEGG (Kyoto En-
cyclopedia of Genes and Genomes) pathway analysis, and
thus this finding could change our concept of pathway anal-
ysis and selection pressure on such cascades. Although it is
unusual for a deleterious biochemical reaction network to de-
velop because of natural selection, the fact that these aber-
rant reactions occur in the setting of neurodegenerative
diseases in aged individuals, far beyond their reproductive
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years, may explain why they are subject to less evolutionary
selection pressure (47).
Our mass spectrometry screening method uncovered a
large number of potentially S-nitrosylated or transnitrosyl-
ated proteins in the brain (table S1). In the future, as mass
spectrometry methods continue to advance, additional mem-
bers of this noncanonical transnitrosylation network and ad-
ditional transnitrosylation pathways in various disease states
may be discovered. In summary, we find that enzymes previ-
ously thought to be in distinct biochemical cascades are ac-
tually linked by pathological crosstalk via transnitrosylation
under disease conditions that may contribute in a causal
manner to the disease process. The transnitrosylation activity
of these enzymes may represent a therapeutic target for AD.
Materials and methods
Cell lines
SH-SY5Y cells (ATCC; CRL-2266) and HEK cells stably ex-
pressing nNOS (HEK-nNOS cells, gift from Drs. David Bredt
and Solomon Snyder) were maintained in DMEM (Thermo
Fisher Scientific) supplemented with 10% fetal bovine serum,
2 mM l-glutamine (Thermo Fisher Scientific), 50 IU/ml peni-
cillin (Omega Scientific), and 50 μg/ml streptomycin (Omega
Scientific). For HEK-nNOS cell culture, 100 μg/ml geneticin
(Thermo Fisher Scientific) was also included in the culture
medium to maintain the expression of nNOS, and nNOS ac-
tivation was achieved by the incubation with the calcium ion-
ophore, A23187 (10 μM, EMD Millipore). Primary rat cortical
neurons were isolated and cultured as described previously
(15, 39, 41, 48).
E. coli strain for purification of recombinant Uch-L1
BL21 (DE3) E. coli (Agilent Technology) were transformed
with a pET28a vector containing His-tagged Uch-L1 (WT or
C152S mutant) and grown in 2XYT medium until the optical
density measured at 600 nm (OD 600) reached 0.5. Expres-
sion of Uch-L1 was induced by 0.5 mM IPTG for 16 hours, and
E. coli were collected by centrifugation. The bacterial pellets
(Page numbers not final at time of first release) 5
were re-suspended in PBS, frozen down at −80°C for 20 min,
and then thawed at room temperature for cell disruption.
Cells were further disrupted by sonication on ice, followed by
incubation in 1% Triton X-100 at 4°C for 30 min. The samples
were centrifuged for 15 min at 10,000 × g, collected as super-
natants, and then centrifuged again to completely remove
bacterial debris. Recombinant Uch-L1 protein was purified
using Ni-NTA agarose (Qiagen) or TALON metal affinity resin
(Takara) at room temperature for 1 hour. Imidazole (50 mM)
was used for protein elution, and excess imidazole removed
by PD-10 columns (GE Healthcare Life Sciences).
Animals and patient brain samples
We used two mouse models of AD (3-6 months old): 1)
Tg2576, overexpressing a mutant form of human (h)APP with
the Swedish mutation (KM670/671NL) (Taconic Biosciences,
Model 1349) (49), and 2) hAPP-J20, expressing hAPP with two
familial AD-linked mutations (the Swedish and Indiana
[V717F] mutations) (The Jackson Laboratory, 34836-JAX)
(42). Age and sex matched samples were randomly chosen
and assigned to different experimental groups. All experi-
ments were performed in accordance with the guidelines out-
lined by the Institutional Animal Care and Use Committees
at The Sanford Burnham Prebys Medical Discovery Institute,
the Scintillon Institute, or The Scripps Research Institute.
Postmortem brain tissues from AD or age-matched con-
trol patients (age <95) were collected at the University of Cal-
ifornia, San Diego, School of Medicine from subjects whose
age, postmortem interval, and gender are described in table
S2. Human brain samples were analyzed with institutional
permission under the state of California and NIH guidelines.
Informed consent was obtained according to procedures ap-
proved by the Institutional Review Board at the University of
California, San Diego, School of Medicine.
NO donors, Aβ oligomer preparation, cell cultures,
plasmids, and transfections
For induction of S-nitrosothiol formation using exogenous
NO donors, the rapidly-decaying NO donor, SNOC was syn-
thesized as described previously (50) and employed for short-
term additions of NO. Oligomerized Aβ1-42 was prepared as we
described previously (36). HEK-nNOS cells and rat primary
neurons were transfected with Lipofectamine 2000 (Thermo
Fisher Scientific) as per the manufacturer’s instruction.
Lipofectamine LTX with PLUS reagent (Thermo Fisher Sci-
entific) was used for transfection of SH-SY5Y cells. Measure-
ments of dendritic spine density in rat primary cortical
neuron cultures were performed in a masked fashion, as we
previously described (15, 36, 48).
For exogenous expression of Uch-L1 in mammalian cells,
the human cDNA sequence for Uch-L1 containing N-terminal
V5 tag or C-terminal myc tag was subcloned into pcDNA3.1
First release: 3 December 2020 www.sciencemag.org
vector (Thermo Fisher Scientific). For knockdown experi-
ments, HEK-293 cells were transfected with siUch-L1 (Am-
bion, #4390824, ID: s14616) or Silencer Select Negative
Control No. 1 siRNA (Ambion, #4390843) using Lipofec-
tamine RNAiMAX Transfection Reagent (Thermo Fisher Sci-
entific) according to the manufacturer’s protocol and
incubated for 72 hours to achieve Uch-L1 knockdown. For
bacterial expression, the Uch-L1 sequence was inserted into
pET28a, adding a His tag to its N terminus. C47S, C90S,
C132S, C152S, C201S, C220S Uch-L1 mutants were generated
using the QuikChange Lightning Multi Site-Directed Muta-
genesis Kit (Agilent Technologies) according to the manufac-
turer’s protocol.
Downloaded from http://science.sciencemag.org/ on December 4, 2020
Biotin-switch assays and immunoblots
For analysis of S-nitrosylated proteins, we performed the bi-
otin-switch assay as previously described (15, 39, 41). In brief,
cell or tissue homogenates were incubated with the MMTS
(S-methyl methanethiosulfonate, Sigma-Aldrich) blocking
buffer (10 mM MMTS, 2.5% SDS in HEN [100 mM Hepes, pH
7.4, 1 mM EDTA, 0.1 mM neocuproine] buffer) to block free
thiol groups. After specific reduction of S-nitrosothiols by 20
mM ascorbate, the newly generated free thiol groups were bi-
otinylated with 1 mM biotin-HPDP (Thermo Fisher Scientific
or Soltec Ventures). For control groups, ascorbate was omit-
ted from the procedure to ensure the specificity of biotinyla-
tion. The biotinylated proteins were pulled down using High
Capacity NeutrAvidin agarose (Thermo Fisher Scientific),
and the purified proteins were subjected to immunoblot anal-
ysis. For the loading control, 5% input was saved before con-
ducting a NeutrAvidin pull-down.
Protein samples were resolved by Bolt Bis-Tris Plus gels
(Thermo Fisher Scientific) and transferred to Immobilon-FL
PVDF membranes (EMD Millipore). After blocking with Od-
yssey TBS blocking buffer (Li-Cor), the membranes were in-
cubated with primary antibodies against Uch-L1 (1:1000,
Sigma-Aldrich, U5258), myc (1:1000, Cell Signaling Technol-
ogy, 2276), V5 (1:5000, Thermo Fisher Scientific, R960-25),
HA (1:1000, Santa Cruz Biotechnology, sc-805), Cdk5 (1:1000,
Santa Cruz Biotechnology, sc-173 or Cell Signaling Technol-
ogy, 12134), or Drp1 (1:1000, BD Biosciences, 611113). IR-dye
680LT-conjugated goat anti-mouse IgG (1:15000, Li-Cor, 926-
68020) and IR-dye 800CW-conjugated goat anti-rabbit IgG
(1:15000, Li-Cor, 926-32211) were used as secondary antibod-
ies. For visualization of target proteins, the membranes were
scanned with an infrared-based Odyssey imaging system (Li-
Cor), and quantification of each band was performed with
Image Studio software (Li-Cor).
Resin-assisted capture of S-nitrosylated proteins
(SNO-RAC) for unbiased mass spectrometry analysis
To purify S-nitrosylated proteins, including SNO-Uch-L1,
(Page numbers not final at time of first release) 6
SNO-RAC was performed as previously described (51) with
minor modifications. In brief, mouse brain cerebrocortical ly-
sates were prepared in cold HENTS (1% Triton X-100, 0.1%
SDS in HEN [100 mM Hepes, pH 7.4, 1 mM EDTA, 0.1 mM
neocuproine]) buffer, assayed for protein concentration us-
ing the Bio-Rad Protein Assay Dye Reagent Concentrate (Bi-
oRad), and diluted with HEN buffer to a concentration of 1
μg/μl. SNOC (final concentration 25 μM) was added to 250 μl
of mouse brain lysate, and the mixture was left at room tem-
perature for 25 min in the dark to allow formation of S-nitro-
sylated (SNO)-proteins. Protein thiols were blocked with
MMTS (50 mM) at 50°C for 30 min, and excess MMTS re-
moved by acetone precipitation. S-Nitrosothiols were then
converted to free thiols with ascorbate (50 mM), and then
captured by thiopropyl sepharose 6B resin (GE Healthcare
Life Sciences) with rotation at room temperature for 2 hours.
The samples were washed twice with HENS buffer (HEN con-
taining 1% SDS), twice with HENS/NaCl (HENS containing
400 mM NaCl), twice with HENS/10 (1:10 HENS), and then
once with trypsin digestion buffer (50 mM NH4HCO3, 1 mM
EDTA). Trypsin digestion was conducted on resin at 37°C for
12 hours, and the eluate was subjected to analysis by mass
spectrometry (MS).
Digested peptide mixtures were pressure-loaded onto a
fused silica capillary column of 250-μm internal diameter
(I.D.) packed with 3 cm of 5 μm Partisphere strong cation ex-
changer (SCX, Whatman) and 3 cm of 10 μm Jupiter reversed-
phase C12 material (Phenomenex); the SCX was end fritted
with immobilized Kasil 1624 (PQ Corp.). After desalting, a 100
μm I.D. capillary with a 5 μm pulled tip packed with 15 cm of
4 μm Jupiter C12 material was attached to a zero-dead-vol-
ume union, and the entire split-column was placed in line
with an Eksigent nano HPLC pump (Eksigent) and analyzed
using modified, four-step multi-dimensional protein identifi-
cation technology (MudPIT), as described previously (52).
The eluted peptides were electrosprayed directly into a hy-
brid LTQ-Orbitrap mass spectrometer (ThermoFinnigan)
with a distal spray voltage of 2.4 kV. A cycle of one full-scan
mass spectrum (400-1400 m/z) followed by 6 data-dependent
MS/MS scans were repeated continuously. Mass spectrum
data were collected using Thermo XCalibur 3.1 (Thermo
Fisher Scientific). The MS/MS spectra were searched with the
ProLucid (a SEQUEST-based software) algorithm (53) against
the EBI mouse IPI database concatenated to a decoy database
in which the sequence for each entry in the original database
was reversed. No modification on cysteine was allowed to re-
flect the reduced SNO thiols. The search results were assem-
bled and filtered using the DTASelect program (54) with a
peptide false-positive rate of 1%. All peptides identified were
at least half tryptic, and precursor ion mass tolerance was set
to 20 parts per million (ppm).
First release: 3 December 2020 www.sciencemag.org
Ubiquitin pull-down assays
To prepare resin-bound SNO-Uch-L1 in vitro, recombinant
Uch-L1 bound to Ni-NTA agarose was mixed with SNOC (100
μM) or “old” SNOC (from which NO had been dissipated), and
left at room temperature for 30 min in the dark with occa-
sional mixing. The samples were washed once with PBS, and
resuspended in PBS containing 0.5% Triton X-100. Recombi-
nant Ubiquitin (Ub) (Sigma) was then mixed with resin-
bound SNO-Uch-L1 (or a reduced form of Uch-L1) and incu-
bated at room temperature for 1 hour with rotation. After in-
cubation, the resin was washed twice with PBS containing
0.5% Triton X-100. The samples were resuspended in Nu-
PAGE LDS sample buffer (Thermo Fisher Scientific), boiled
at 95°C for 3 min, and loaded onto NuPAGE Bis-Tris gel. The
Downloaded from http://science.sciencemag.org/ on December 4, 2020
bands were visualized by the standard Coomassie staining
method.
Uch-L1 deubiquitinylation activity assays
Deubiquitinylation activity of recombinant Uch-L1 was fluo-
rometrically assayed using Ub-7-amido-4-methylcoumarin
(Ub-AMC, Boston Biochem) as described (29) with minor
modification. In brief, 0.25 μM Ub-AMC and 0.02 μM Uch-L1
(or SNO-Uch-L1) were mixed in a final volume of 100 μl in
reaction buffer (50 mM Tris-HCl, pH 7.6, 0.5 mM EDTA), and
incubated for 10 min at 25°C. The amount of AMC released
from the Ub-AMC substrate was monitored using SpectraMax
M2 (Molecular Devices) with excitation and emission wave-
lengths of 380 nm and 460 nm, respectively.
Activity of Uch-L1 in cells or tissue lysates was assessed
with the activity-based probe, ubiquitin-vinylmethyl ester
(Ub-VME), irreversibly modifying the “active” form of ubiq-
uitin C‐terminal hydrolases and thus causing a shift in elec-
trophoretic mobility on immunoblots (55, 56). For the Ub-
VME assay, cell or tissue lysate was freshly prepared in PBS
containing 0.1% Triton X-100. The reaction was initiated by
incubating 2 μg of lysate with 0.2 μM Ub-VME (Boston Bio-
chem), incubated for 5 min at room temperature, and
stopped by adding NuPAGE LDS sample buffer (Thermo
Fisher Scientific). Proteins were resolved by NuPAGE Bis-Tris
gel followed by Western blotting.
Lentivirus production, stereotaxic injection, and
quantitative immunohistochemistry
Uch-L1 (WT or C152S mutant) was subcloned into lentiviral
pCDH-EF1-dTomato-T2A-Kpn21 (System Biosciences) and
used for preparation of active lentiviral particles for animal
injections (57). hAPP-J20 AD transgenic mice or non-trans-
genic littermate control mice at the age of 3-4 months re-
ceived bilateral, stereotactic injections of lentiviral vectors
into the dentate gyrus at a rate of 1 μl per 2 min for a total of
2 μl of lentivirus infused into each hemisphere. Coordinates
used for viral injection were as follows (35): AP-2.1, ML±1.8,
(Page numbers not final at time of first release) 7
and DV-2.0. Eight weeks after lentiviral injection, mice were
anesthetized, perfused transcardially with saline (0.9% NaCl)
containing 20 mU/ml Heparin (Sigma), followed by 4% PFA
perfusion. Brains were then removed and fixed in 4% PFA for
48 hours at 4°C and kept in 1% PFA until analyzed. Quantita-
tive immunohistochemistry was performed as described (36,
37). Immunolabeling was performed using mouse monoclo-
nal antibody against Synaptophysin (Clone SY38, Millipore).
Analysis of co-localization was performed on the whole den-
tate gyrus image using 3 separate images per condition by Fiji
(ImageJ) software using the Squassh plugin as previously de-
scribed (58, 59). All procedures described here were approved
by the Institutional Animal Care and Use Committees at the
Scintillon Institute and The Scripps Research Institute,
where the work was performed.
Transnitrosylation assays
SH-SY5Y cells were transfected with Uch-L1-V5 or Cdk5-HA.
After 1 day, Uch-L1-V5 expressing cells were lysed in RIPA
buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1% Nonidet
P-40, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate) and
immunoprecipitated overnight at 4°C with anti-V5 antibody
conjugated to Dynabeads Protein G (Thermo Fisher Scien-
tific), and then washed 3 times with high-salt RIPA buffer (50
mM Tris–HCl, pH 8.0, 500 mM NaCl, 1% Nonidet P-40, 0.5%
deoxycholate, 0.1% sodium dodecyl sulfate). Immunoprecipi-
tated Uch-L1-V5 was exposed to decayed or freshly prepared
100 μM SNOC in lysis buffer (PBS containing 0.1% Triton X-
100) for 30 min at room temperature in the dark with gentle
shaking. Concurrently, cell lysates were prepared in lysis buffer
(PBS containing 1% Triton X-100) from SH-SY5Y cells express-
ing Cdk5-HA. Immunoprecipitated Uch-L1-V5 exposed to
SNOC (or as a control “old” SNOC, from which NO had been
dissipated) was then added to SH-SY5Y whole-cell lysates and
incubated for 1 hour at room temperature in the dark with gen-
tle shaking. These reaction mixtures were subsequently sub-
jected to biotin-switch assay after addition of MMTS blocking
buffer. Dynabeads used for immunoprecipitation were re-
moved by magnet rack prior to acetone precipitation.
For transnitrosylation assays using Cdk5-immuno-
depleted cell lysates, SH-SY5Y cells were transfected with WT
Uch-L1-V5, mutant Uch-L1(C152S)-V5, or Drp1-HA. After 1
day, Uch-L1-V5—expressing cells were lysed in lysis buffer
(PBS containing 1% Triton X-100) and immunoprecipitated
with anti-V5 antibody. SH-SY5Y cells expressing Drp1-HA
were also lysed in lysis buffer and immunodepleted overnight
at 4°C with anti-Cdk5 antibody (C-8, Santa Cruz Biotechnol-
ogy, sc-173); or rabbit IgG (Santa Cruz, SC-2027) was used as
a control antibody. Immunoprecipitated Uch-L1-V5 was
washed in high-salt RIPA buffer (50 mM Tris–HCl, pH 8.0,
500 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% so-
dium dodecyl sulfate) and exposed to freshly prepared or
First release: 3 December 2020 www.sciencemag.org
“old” SNOC and then mixed with SH-SY5Y cell lysates immu-
nodepleted of Cdk5. Transnitrosylation was assessed by bio-
tin-switch assay. To calculate the redox potential and change
in Gibbs free energy for the transnitrosylation reaction, Uch-
L1-V5 and CDK5-HA co-transfected SH-SY5Y cells were ex-
posed to 50 μM SNOC; after 30 min, cell lysates were sub-
jected to biotin-switch assay in the absence or presence of
MMTS free-thiol blocking agent, allowing us to observe total
protein (both the reduced and S-nitrosylated forms) and oxi-
dized protein (the S-nitrosylated forms), respectively. Subse-
quently, immunoblotted bands were quantified by
densitometry, and the values used in a ratio of Nernst equa-
tions, as described in the manuscript text.
For in vitro transnitrosylation assays with recombinant
Downloaded from http://science.sciencemag.org/ on December 4, 2020
proteins to detect direct transfer of the NO group from one
protein to another, we first exposed purified Uch-L1 (10 μM)
to 200 μM SNOC and then incubated for 30 min to generate
SNO-Uch-L1 while allowing NO to dissipate from SNOC. Re-
sidual SNOC or cysteine remaining after NO dissipation was
removed with a Zeba Spin Desalting Column (Thermo Fisher
Scientific), and the resulting purified SNO-Uch-L1 was incu-
bated with recombinant Cdk5/p25 (2 μM, Abcam, ab60761)
for an additional 45 min to facilitate transnitrosylation from
Uch-L1 to Cdk5. Note that p25 was included for stabilization
of Cdk5, as we and others had previously shown that Cdk5
was the S-nitrosylated species (19). The biotin-switch assay
was then performed as described above.
Quantification and statistical analysis
Fiji (ImageJ) and Image Studio were used for quantification
of immunohistochemistry and immunoblot data, respec-
tively. A Power Analysis of our prior data was used to deter-
mine the number of replicates needed for statistical purposes.
The number of replicates or experiments are indicated in the
individual figure legends. Data are expressed as mean + SEM.
Differences between experimental groups were evaluated us-
ing an ANOVA followed by a post hoc test for multiple com-
parisons (Dunnett’s or Bonferroni’s) or a Student’s t test for
comparison between two groups. A P value  < 0.05 was con-
sidered significant. Statistical analyses were performed using
GraphPad Prism software.
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ACKNOWLEDGMENTS
We thank Drs. David Bredt and Solomon Snyder (Johns Hopkins) for HEK-nNOS
cells, Traci Fang-Newmeyer (Sanford Burnham Prebys Medical Discovery
Institute) and Weiping Tan (The Scripps Research Institute) for preparing
cultures, and Scott R. McKercher (The Scripps Research Institute) for help with
manuscript critical review and preparation. Funding: This work was supported in
part by NIH grants RF1 AG057409, R01 AG056259, R01 DA048882, R01
NS086890, P30 NS076411, P01 ES016738 and DP1 DA041722 (to S.A.L.), R01
AG061845 (to T.N.), P30 AG062429 and R01 AG018440 (to R.A.R), and P41
GM103533 (to J.R.Y.); by a Distinguished Investigator Award from the Brain &
Behavior Research Foundation (to S.A.L.); by a New Investigator Award from the
Alzheimer’s Association (to T.N.); by the Michael J. Fox Foundation (to S.A.L. and
T.N.); and by the Step Family Foundation (to S.A.L.). Author contributions:
S.A.L. conceived, designed, and supervised the execution of the entire project.
T.N., C.O., and S.A.L. formulated the detailed research plans, interpreted
experimental results, and wrote the manuscript. T.N. and C.O. performed a
majority of molecular and biochemical experiments, X.Z. carried out biochemical
experiments with the J20 mouse model of AD, and A.K.D. and H.S. conducted
some biochemical experiments with cell culture models. L.L. and J.R.Y. were
responsible for mass spectrometry analysis, K.M.L. performed lentiviral
injections into mouse brains, and D.G. prepared lentiviral constructs for the
study. B.S., E.M., and R.S. were responsible for histological analysis. Competing
interests: D.G. is currently Director of Vector Development at LocanaBio, Inc.
(San Diego, California). Data and materials availability: All data are available in
the main text or the supplementary materials. All plasmids generated in this
study are available from Stuart A. Lipton under a material transfer agreement
with The Scripps Research Institute.
SUPPLEMENTARY MATERIALS
science.sciencemag.org/cgi/content/full/science.aaw0843/DC1
Figs. S1 to S7
Tables S1 and S2
MDAR Reproducibility Checklist
15 November 2018; accepted 18 November 2020
Published online 3 December 2020
10.1126/science.aaw0843
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Fig. 1. S-Nitrosylation of Uch-L1 at Cys152. (A) SNO-Uch-L1 formation by biotin-switch assay in SH-SY5Y cells
exposed to 100 μM SNOC. As a negative control, ascorbate (Asc) was omitted. Values are expressed as mean
+ SEM (n = 3 biological replicates per group). **P < 0.01 by ANOVA. (B) SNO-Uch-L1 formation by biotin-switch
assay in HEK-nNOS cells exposed to calcium ionophore A23187. (C) Presence of SNO-Uch-L1 in the
cerebrocortex of 3-6 mos-old hAPP-J20 AD transgenic mice by biotin-switch assay. Values are mean + SEM (n
= 6 per group). *P < 0.05 by Student’s t test. (D) S-Nitrosylation of Uch-L1 at Cys152. HEK cells expressing myc-
tagged WT Uch-L1 or cysteine mutants of Uch-L1 were exposed to SNOC. Lack of a band by biotin-switch assay
for Uch-L1(C152S) indicates that it is the predominant site of S-nitrosylation. Values are expressed as mean +
SEM (n = 3-4 per group). *P < 0.05 compared to “WT Uch-L1 + SNOC” by ANOVA. (E) S-Nitrosylation of Uch-
L1 at Cys152 by biotin-switch assay in HEK-nNOS cells. HEK-nNOS cells expressing myc-tagged Uch-L1 WT or
C152S mutant were exposed to A23187 to increase intracellular Ca2+ and thus activate nNOS to generate
endogenous NO. The NOS inhibitor NG-nitro-l-arginine (NNA, 1 mM) was used to inhibit NO generation as a
control. Values are expressed as mean + SEM (n = 3 per group). *P < 0.05 by ANOVA. Uncropped immunoblots
are included in fig. S7.

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Fig. 2. Mass spectrometry (MS) identification of S-nitrosylated Cys
residue in Uch-L1 and atomic resolution model. (A) Table summarizing S-
nitrosylated peptides for Uch-L1 by MS analysis. Four peptides were
identified containing Cys152, and one peptide with Cys220. Spectral counting
clearly demonstrated that Cys152 is the predominant S-nitrosylation site on
Uch-L1 (total spectral counts 33 for Cys152 vs. 2 for Cys220). (B) Tandem
mass spectrum of a Uchl-L1 peptide identifying S-nitrosylation at Cys152. The
measured mass, charge state, and measurement accuracy of the precursor
ion are displayed. The b (blue) and y (red) fragment ions are annotated with
the measured mass. (C) Crystal structure of Uch-L1 (PDB ID: 2ETL) near
Cys152 (yellow). Magenta: Glu7 and Arg153. Molecular visualization and
graphics handling were performed using PyMol.

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Fig. 3. Aβ-induced S-nitrosylation of Uch-L1 contributes to AD-related synapse loss. (A) SNO-Uch-L1 formation
in primary rat cerebrocortical neurons in culture after 4.5 hours exposure to 500 nM oligomerized Aβ1-42 assayed by
the biotin-switch method (upper panel). Quantification of biotin-switch blots (lower panel). Values are mean + SEM
(n = 3 per group). *P < 0.05 by Student’s t test. (B and C) Exposure of rat cerebrocortical neurons in vitro to Aβ
oligomers (500 nM) for 24 hours resulted in decreased dendritic spines, visualized by GFP transfection (B).
Transfection with non-nitrosylatable mutant Uch-L1(C152S) abrogated Aβ-induced dendritic spine damage in a
statistically significant manner, while WT Uch-L1 manifested a lesser, nonsignificant effect. Values are mean + SEM
(spines scored for n = 25 neurons). *P < 0.05 by ANOVA with Bonferroni correction. (D and E) Lentiviral expression
of Uch-L1(C152S) in vivo prevents loss of presynaptic marker in hAPP-J20 mice. Representative immunostaining of
hippocampal sections with synaptophysin antibody (SY38; pseudo-colored red). Scale bar, 200 μm (D).
Quantification of hippocampal immunohistochemistry from WT and hAPP-J20 mice injected with WT Uch-L1, mutant
Uch-L1(C152S), or empty lentiviral vectors (E). Immunohistochemistry was performed with synaptophysin antibody
(SY38). Values are mean + SEM (n = 18 mice injected). *P < 0.05, ***P < 0.001 by Student’s t test with Bonferroni
correction.
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Fig. 4. 4. Transnitrosylation from SNO-Uch-L1 to Cdk5 on biotin-
switch assays. (A) SNO-Uch-L1 formed 10-min after exposure of HEK-
293 cells to 100 μM SNOC, but not after exposure to control
represented by “old” SNOC from which the NO group had been
dissipated, although this solution might still contain nitrite and
disulfides. Formation of SNO-Cdk5 was also observed, and this was
partially abrogated by siRNA knockdown of Uch-L1 (siUch-L1)
compared to control siRNA (siCTL) cells. (B) Knockdown of Uch-L1
largely prevented formation of SNO-Drp1, which first appeared 25 min
after exposure to SNOC. (C) Quantification of SNO-Cdk5 on biotin-
switch blots. Relative SNO-Cdk5 levels (SNO-protein/Input protein) in
siCTL cells were increased 10 min after exposure to SNOC vs. 25 min
when compared to siUch-L1 cells exposed to SNOC. Values are mean +
SEM (n = 3 per group). ***P < 0.001 by two-tailed Student’s t test. (D)
Similar type of quantification of SNO-Drp1 on biotin-switch blots. In this
case, relative SNO-Drp1 levels were increased 25 min after exposure to
SNOC vs. 10 min. Values are mean + SEM (n = 3 per group). ***P <
0.001 by two-tailed Student’s t test.

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 Fig. 5. Triple transnitrosylation from
SNO-Uch-L1 to SNO-Cdk5 to SNO-
Drp1. (A) Schematic diagram of in
vitro transnitrosylation assay. (B)
Transnitrosylation occurs from WT
SNO-Uch-L1 to Cdk5 to Drp1 but
significantly less with mutant Uch-
L1(C152S). WT Uch-L1-V5 or mutant
Uch-L1(C152S)-V5 from SH-SY5Y
cell lysates was immunoprecipitated
with anti-V5 antibody and exposed to
100 μM SNOC. These
immunoprecipitates were then added
to whole cell lysates expressing HA-
Cdk5, which were subjected to the
biotin-switch assay to assess

Downloaded from http://science.sciencemag.org/ on December 4, 2020

transnitrosylation. Compilation of
representative blots shown. (C and
D) Quantification of Uch-L1 and Cdk5
biotin-switch blots. Values are mean
+ SEM (n = 4 per group). ***P < 0.001
by two-tailed Student’s t test, **P <
0.01, *P < 0.05 by ANOVA. (E)
Quantification of Drp1 biotin-switch
blots. Values are mean + SEM (n = 3
per group). *P < 0.05, **P < 0.01 by
ANOVA. (F) Schematic diagram of in
vitro transnitrosylation assay after
immunoprecipitation to decrease the
amount of endogenous Cdk5. (G)
SNO-Uch-L1 nitrosylation of Drp1 is
affected by the level of endogenous
Cdk5. WT Uch-L1-V5 or Uch-
L1(C152S)-V5 from SH-SY5Y cell
lysates was immunoprecipitated with
anti-V5 antibody, and the
immunoprecipitates exposed to 100
μM SNOC or old SNOC. The
immunoprecipitates containing
SNOC-exposed Uch-L1 proteins were
then incubated with SH-SY5Y cell
lysates expressing Drp1-HA after
substantial immunodepletion of
endogenous Cdk5 with anti-Cdk5
antibody (or IgG control antibody).
Samples were subjected to biotin-
switch assay to assess
transnitrosylation. (H and I)
Quantification of Uch-L1and Drp1
biotin-switch blots. Values are mean
+ SEM (n = 3 per group). **P < 0.01,
*P < 0.05 by ANOVA.

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 Downloaded from http://science.sciencemag.org/ on December 4, 2020
Fig. 6. Redox blot quantifying transnitrosylation from Uch-L1 to Cdk5. (A)
Transnitrosylation occurs predominantly from SNO-Uch-L1 to Cdk5 to Drp1
rather than in the opposite direction from SNO-Cdk5 to Uch-L1. Expression of
Uch-L1-V5 and Cdk5-HA increased the formation of SNO-Drp1, as evidenced
on biotin-switch assay. Lysates of SH-SY5Y cells were prepared by V5- or HA-
immunoprecipitation and exposed to 100 μM SNOC. These
immunoprecipitates were then added to whole-cell lysates expressing either
HA-Cdk5 or Uch-L1-V5, which were subjected to the biotin-switch assay to
assess transnitrosylation. (B) SH-SY5Y cells were transfected with Uchl-L1-V5
and Cdk5-HA tagged constructs, and exposed to 50 μM SNOC at room
temperature. After 30 min, cell lysates were prepared, incubated to achieve
steady state (~1 hour, by which time SNOC, a short-lived NO donor, had
completely dissipated), and then subjected to the biotin-switch assay. Methyl-
methanethiosulfonate (MMTS, 25 mM) was used to block free thiols during
the assay for S-nitrosylated protein. Chemically-reduced thiol protein
represents the relative amount of total protein obtained by the biotin-switch
assay performed in the absence of MMTS (w/o MMTS). SNO-protein
represents the relative amount of S-nitrosylated protein obtained by the
biotin-switch assay. The relative concentration of protein in a redox pair in the
S-nitrosylated (oxidized) form and the reduced form can be measured by
quantitative densitometry of their respective bands on the gels (n = 4).

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 Downloaded from http://science.sciencemag.org/ on December 4, 2020
Fig. 7. S-Nitrosylation of Uch-L1 in human AD brains and schema of transnitrosylation in the
pathophysiology of synapse loss in AD. (A) Human brain tissues from control or AD patients were
subjected to the biotin-switch assay to detect SNO-Uch-L1. (B) Quantification of the ratio of SNO-
Uch-L1 to total Uch-L1 in human brains and in cell-based assays. Biotin-switch assays and
immunoblot analyses were quantified by densitometry, and the relative ratio of SNO-Uch-L1 to total
Uch-L1 was calculated for the following conditions: In vitro in cerebrocortical neurons after Aβ
exposure (as shown in Fig. 3A), in vivo in the hAPP-J20 mouse model of AD (as shown in Fig. 1C),
and in human AD brains vs. control human brains. Values are mean + SEM (n = 6 for control human
brains, n = 7 samples from 5 AD human brains, n = 3 for each group in primary neuron experiments,
n = 6 for each group in mouse brain experiments). ***P < 0.001, *P < 0.05 by Student’s t test. (C)
Biochemical schema of transnitrosylation pathway leading to synaptic damage and consequent
memory loss in AD. Note that these transnitrosylation reactions may be direct or indirect, with
additional, as yet unknown, members of the transnitrosylation network still to be discovered.

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Noncanonical transnitrosylation network contributes to synapse loss in Alzheimer's disease
Tomohiro Nakamura, Chang-ki Oh, Lujian Liao, Xu Zhang, Kevin M. Lopez, Daniel Gibbs, Amanda K. Deal, Henry R. Scott, Brian
Spencer, Eliezer Masliah, Robert A. Rissman, John R. Yates III and Stuart A. Lipton

published online December 3, 2020

Downloaded from http://science.sciencemag.org/ on December 4, 2020

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